Kits for detecting breast or ovarian cancer in a body fluid sample and use thereof

ABSTRACT

The present invention relates to a method for the accurate, rapid and sensitive detection of breast or ovarian cancers from body fluid samples of a mammalian subject and related assay, kits and peptides suitable for such a method.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/IB2011/054194, filed Sep. 23, 2011.

SEQUENCE LISTING

The present application includes a Sequence Listing in electronicformat. The Sequence Listing is entitled 4472103SequenceListingRev.txt,was created on 19 Mar. 2018 and is 5 kb in size. The information in theelectronic format of the Sequence Listing is part of the presentapplication and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to kits for detection of ovarian andbreast cancers in a sample. In particular, the invention relates to kitsand a method using those kits for the early diagnosis of breast andovarian cancers in biological fluids such as human serum.

BACKGROUND OF THE INVENTION

Breast cancer is the most commonly diagnosed cancer in women afternon-melanoma skin cancer, and is the second leading cause of cancerdeaths after lung cancer. Clinical evaluations have established thatmammography screening with or without clinical breast examination, maydecrease breast cancer mortality. Therefore, mammography remains thegold reference standard today for breast cancer screening, however, itsuffers from a variety of limitations such as radiation hazard,considerable patient discomfort, higher false readouts with denserbreast tissue, sensitivity & specificity varies according totechnician's skill (60-80%), and typically over-diagnoses. Currentlyused serum/blood biomarkers (e.g. CA27.29, CA15.3, CEA, HER-2) aremainly used in monitoring and surveillance due to poor sensitivity andspecificity for other than those applications. Patient managementfollowing initial suspicion of breast cancer usually includesconfirmation of the diagnosis, evaluation of stage of disease, surgicalremoval of the tumour tissue and selection of a therapy. The survivalrate to breast cancer is over 90% when detected and treated early withexisting therapies.

Ovarian cancer is the second most common gynaecological cancer and isthe ninth most common cancer but is the fifth most deadly. In the USA,ovarian cancer occurs in 1 of 2′500 post-menopausal women and is themost lethal gynaecological malignancy, accounting for 5-6% of allcancer-related deaths (Kulasingam et al., 2010, Nature Reviews, Cancer,10, 371-377). The main problem is that ovarian cancer does not usuallylead to alarming symptoms. A large majority of patients have advancedcancer and widespread disease at presentation. This late detection ofovarian cancer explains why the overall survival rate for ovarian canceris less than 40%, whereas that the survival rate is close to 90% whendiagnosed at early stage. Furthermore, there is no effective screeningtest that can detect the disease in its early stages. For women atincreased risk, prophylactic oophorectomy is usually considered afterthe age of 35 if childbearing is complete. Patients with clear cellhistology appear to have a worse prognosis.

The most studied/used marker for ovarian cancer is CA125 which is aprotein antigen found at abnormally high levels in the blood of manyovarian cancer patients, but was also found to be elevated in non-cancercases (e.g. pregnant women). Furthermore, the CA125 biomarker was foundto have a low sensitivity and specificity and is essentially used formonitoring response to treatment for ovarian cancer. Although CA125, hasno prognostic significance when measured at the time of diagnosis, itseems to show a correlation with survival with the progression of thedisease, especially when measured after the course of chemotherapy forpatients with stage III or stage IV disease. Another method used forearly detection of ovarian cancer is trans-vaginal ultrasound. However,this method is known to have a very low positive predictive value ofabout 3% according to a major recent study (Menon et al. 2009, LancetOncol., 10, 327-40).

In most families affected with the breast and ovarian cancer syndrome orsite-specific ovarian cancer, genetic linkage has been found to theBRCA1 locus on chromosome 17q21 (Easton et al., 1993, Am. J. Hum.Genet., 52 (4): 678-701). BRCA2, responsible for some forms of inheritedovarian and breast cancer, has been mapped by genetic linkage tochromosome 13q12 (Wooster et al., 1994, Science 265 (5181): 2088-90,1994). The majority of those women with those mutations presumably havefamily members with a history of ovarian and/or breast cancer;therefore, they may have be more vigilant and inclined to participate incancer screening programs that may have led to earlier detection.However, for other women populations, early detection remains moreuncertain.

Several tests using biomarkers in blood have attempted to make thediagnosis of ovarian (OC) and breast cancer (BC) less invasive, moreaccurate, less invasive or less risky than the use of known diagnostictools, i.e. mammography for BC. However most of them have been discardedafter a series of unfortunate misinterpretations or due to insufficientexperimental evidence (Kulasingam et al., 2010, above).

Therefore, there is an emerging need for developing new methods andtools for providing an easy, reliable (minimum sensitivity andspecificity), non-invasive and early diagnosis of breast and/or ovariancancers, before those cancer cause symptoms.

SUMMARY OF THE INVENTION

The present invention is directed towards to a method for detectingbreast or ovarian cancers from a biological fluid sample of a mammaliansubject and related assay, kits and peptides suitable for the detectionof breast or ovarian cancers. In particular, the invention relates tothe unexpected finding that peptides according to the invention used asantigens adsorbed on a solid surface allow the detection of endogenousantibodies in a blood sample from a subject, which are indicative of abreast or an ovarian cancer.

A first aspect of the invention provides a method for detecting a breastor an ovarian cancer from a biological fluid sample of a mammaliansubject comprising the steps of:

-   -   (a) providing a biological fluid sample from a mammalian        subject;    -   (b) bringing the said biological fluid sample into contact with        a solid matrix where at least one peptide is bound to, wherein        the contacting is under conditions sufficient for binding an        antibody present in the said biological fluid sample to the said        at least one peptide through antigen-antibody interactions and        wherein the said at least one peptide has a sequence of amino        acids of a peptide according to the invention or of any variant        thereof;    -   (c) Removing the biological fluid sample for removing from the        solid matrix any unbound antibody from the surface of the said        solid matrix;    -   (d) Detecting the presence of an antigen-antibody complex bound        to the said solid matrix, wherein the presence of the said        complex is indicative that the biological fluid sample contains        one or more breast or ovarian-cancer associated endogenous        antibodies.

A second aspect of the invention provides a kit for detecting abiomarker for a breast or an ovarian cancer in a biological fluidsample, the kit comprising at least one peptide according to theinvention or at least one variant thereof or a combination thereof.

A third aspect of the invention provides an isolated peptide accordingto the invention.

A fourth aspect of the invention provides an immunoassay preparation forthe detection of a breast or ovarian cancer comprising at least onepeptide according to the invention.

A fifth aspect of the invention provides a use of a peptide according tothe invention in an assay for the detection of a breast or ovariancancer.

A sixth aspect of the invention provides a use of a peptide according tothe invention or of an immunoassay preparation according to theinvention for the coating of a solid matrix for performing animmunoassay.

A seventh aspect of the invention resides in a use of a kit according tothe invention for detecting a breast or an ovarian cancer from abiological fluid sample of a mammalian subject.

Other features and advantages of the invention will be apparent from thedetailed description, figures and sequences.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the optical density (OD) measured at 405 nm from an inverseELISA assay from one of the studies as described in Example 2 onthirteen blood samples of ovarian cancer (FIG. 1A) and seven controls,i.e. healthy samples (FIG. 1B) as described in Example 3. The x-axisrepresents the peptides used in the assay which are detailed in Table 2,as well as the notation identifying the samples. Boxes represent thevariance of 3 measurements. The vertical lines divide the graph bysample and the horizontal line at OD_(40.5 nm)=0.4 allows bettervisualization of the higher response from cancer samples versuscontrols. Note that in this study it was indeed possible to distinguishthose patients in remission, as indicated in FIG. 1A (samples 11-13). InFIG. 1B, one sample (control 7) showed a significantly higher responsethan other control cases studies. It was unclear ultimately if this wasdue to an error or if indeed the corresponding control person hadcancer. FIG. 1C shows results from four ovarian cancer cases (of whichone was a remission case: sample 3) and four healthy controls that werecarried out by an independent professional diagnostics laboratory.Overall the results were reproducible, with the remission case againclearly discernable.

FIG. 2 shows results obtained from the testing set with the LinearDiscreet Analysis (LDA) classification method (Friedman, 1989,Regularized Discriminant Analysis In: Journal of the AmericanStatistical Association, 84(405): 165-175) on the breast cancer testingset as described in Example 3. The LDA scores obtained lie in the rangeof −5 to +3 approximately, as illustrated in FIGS. 2A & 2B. BBV=BenignBreast cyst (control); BOV=Benign Ovarian Cyst (control); GVABB=Healthyblood donors (control); CBV=Malignant Breast tumour (Cancer cases). InFIG. 2A, the LDA scores of the controls are grouped separately fromthose of breast cancer cases. In FIG. 2B, the scores of each samplegroup are shown separately, in order to allow to see how well theresults/invention can separate malignant from benign tissue. FIG. 2Cshows the Receiver Operating Curve (ROC) of results from the testingset. The ROC is obtained by plotting the sensitivity (or true positiverate) versus false positive rate (1-specificity) for a binaryclassification system (e.g. sick or healthy), as its discriminationthreshold is varied. The area under the curve (AUC) of the ROC is areflection of the performance of the binary classification system ortest. A perfect test would result in an AUC=1. The ROC was plotted byvarying the threshold (LDA score) as described in Example 3. Byselecting a certain threshold LDA score, above which the result isconsidered positive (i.e., presence of cancer), the sensitivity wasplotted (true positive rate) versus (1—specificity) leading to anAUC=0.92.

DETAILED DESCRIPTION OF THE INVENTION

The term “subject” as used herein refers to mammals. For examples,mammals contemplated by the present invention include human, primates,domesticated animals such as cattle, sheep, pigs, horses and the like.

The term “isolated” is used to indicate that the molecule is free ofassociation with other proteins or polypeptides, for example as apurification product.

The expression “breast cancer” includes malignant breast neoplasm, i.e.cancer originating from breast tissue, most commonly from the innerlining of milk ducts or the lobules that supply the ducts with milk.

The expression “ovarian cancer” includes any of various malignantneoplasms characterized by the proliferation of anaplastic cells thattend to invade surrounding tissue of ovaries and metastasize to new bodysites. The pathological condition is characterized by such growths. Forexample, ovarian cancer includes ovarian clear cell carcinoma.

The expression “biological fluid sample” refers to a clinical fluidsample for testing which is taken from a body fluid from a mammal suchas saliva, blood and urine. For example, a biological fluid sample is aserum sample from a human subject.

The expression “control sample” refers to a positive control or anegative control sample. A negative control sample includes a body fluidsample taken from a subject that is the same or homologous species asthe subject to be assayed for autoantibodies and is known to have normalbiological state, e.g. without detectable autoantibodies against atleast one peptide according to the invention or a solution which doesnot contain antibodies that are immunoreactive with at least one peptideaccording to the invention. A negative control sample includes a sampletaken from a control subject. A positive control sample includes a bodyfluid sample taken from a subject that is the same or homologous speciesas the subject to be assayed for autoantibodies and is known to havedetectable autoantibodies against at least one peptide according to theinvention or a solution which does contain antibodies that areimmunoreactive with at least one peptide according to the invention.

The term “variant” as referred to herein, means a polypeptidesubstantially homologous to the original peptide sequence, but which hasat least one an amino acid sequence different from that of the originalsequence because of one or more deletions, insertions or substitutions.Substantially homologous means a variant amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical to the original aminoacid sequences, as disclosed above. The percent identity of two aminoacid sequences can be determined by visual inspection and/ormathematical calculation, or more easily by comparing sequenceinformation using known computer program used for sequence comparisonsuch as Clustal package version 1.83. A variant may comprise a sequencehaving at least one conservatively substituted amino acid, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Generally, substitutions for one or moreamino acids present in the original polypeptide should be madeconservatively. Examples of conservative substitutions includesubstitution of one aliphatic residue for another, such as Ile, Val,Leu, or Ala for one another, or substitutions of one polar residue foranother, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Othersuch conservative substitutions, for example, substitutions of entireregions having similar hydrophobicity characteristics, are well known(Kyte, et al, 1982, J. Mol. Biol., 157: 105-131). For example, a“conservative amino acid substitution” may involve a substitution of anative amino acid residue with a non native residue such that there islittle or no effect on the polarity or charge of the amino acid residueat that position. Desired amino acid substitutions (whether conservativeor non-conservative) can be determined by those skilled in the art atthe time such substitutions are desired. Exemplary amino acidsubstitutions are presented in Table 1 below:

TABLE 1 Original residues Examples of substitutions Ala (A) Val, Leu,Ile Arg (R) Lys, Gln, Asn Asn (N) Gln Asp (D) Glu Cys (C) Ser, Ala GIn(Q) Asn Glu (E) Asp Gly (G) Pro, Ala His (H) Asn, GIn, Lys, Arg Ile (I)Leu, Val, Met, Ala, Phe, Norleucine Leu (L) Ile, Val, Met, Ala, Phe,Norleucine Lys (K) Arg, GIn, Asn Met (M) Leu, Ile, Phe Phe (F) Leu, Val,Ile, Ala, Tyr Pro (P) Ala, Gly Ser (S) Thr, Ala, Cys Thr (T) Ser Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile Met, Leu, Phe, Ala, Norleucine

The term “solid matrix” includes any solid phase support suitable forcarrying out an immunoassay or a method according to the invention. Itincludes beads, microparticles, nanoparticles, tubes, fabrics or plates,films, slides, wells, formed from or coated with glass, polystyrene,polypropylene, nitrocellulose, quartz, ceramic, dextran or othermaterials. For example, the solid matrix is in a form of microtiterwells, such as a 96-well microtiter plate.

The expression “kit” comprises at least one polypeptide according to theinvention or a variant thereof or a combination thereof as describedherein to be coupled or already coupled to a solid matrix and optionallyinstructional material.

Peptides

According to one aspect of the invention, is provided an isolatedpeptide having at least 80% identity or homology with a sequence ofamino acids selected from the group consisting of: SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQID NO: 12.

In a further embodiment, is provided an isolated peptide having at least90% identity or homology with a sequence of amino acids selected fromthe group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

In another further embodiment, is provided an isolated peptide accordingto the invention having an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

Table 2 below presents the Sequence identity numbers and associatedmolecules:

TABLE 2 SEQ ID NO. Molecule 1(p2) EGGTMPDNRQPRNR(C) 2(p8)ILSRKPKPDSDVTQ(C)  3(p11) (C)SVMNTGQRRDGPL  4(p13) VAYHARPDSDQRF(C) 5(p16) DNELSDLKEDKPRK(C)  6(p21) PVCYTPAWIQDLKINRQLDSMIQL(C)  7(p24)KAGRCRIIG(C)  8(p25) MVAVPGPTVAPR(C)  9(p34) LRCSRCNIFG(C) 10(p35)AARVGVKACL(C) 11(p36) DNELSGVKA(C) 12(p37) HIFCSNIFGL(C)

Synthetic chemistry methods, such as solid-phase peptide synthesis, canbe used to synthesize the polypeptides according to the invention.Purification of those peptides may be carried out by means of anytechnique known in the art of protein/peptide purification. Exemplarytechniques include ion-exchange chromatography, hydrophobic interactionchromatography, and immunoaffinity methods.

According to another embodiment, is provided an immunoassay preparationuseful for the detection of a biomarker for a breast or an ovariancancer in a biological fluid sample comprising at least one peptideaccording to the invention.

According to a further embodiment, is provided an immunoassaypreparation useful for the detection of a biomarker for a breast cancerin a biological fluid sample comprising a combination of peptidesaccording to the invention or of variants thereof. In particular, thecombination comprises: a) at least one peptide selected from SEQ ID NO:9 or SEQ ID NO: 12 or a variant thereof; and b) at least one peptideselected from SEQ ID NO: 5 or of SEQ ID NO: 10 or a variant thereof.

According to another further embodiment, is provided an immunoassaypreparation useful for the detection of a biomarker for an ovariancancer in a biological fluid sample a combination of peptides accordingto the invention or of variants thereof. In particular, the combinationcomprises: a) at least one peptide selected from SEQ ID NO: 9 or SEQ IDNO: 12 or a variant thereof; and b) at least one peptide selected fromSEQ ID NO: 2 or of SEQ ID NO: 4 or a variant thereof.

According to another embodiment, is provided a use of an immunoassaypreparation according to the invention for the coating of a solid matrixfor performing an immunoassay.

Kit

According to another aspect of the invention, is provided a kit fordetecting a biomarker for a breast or an ovarian cancer in a biologicalfluid sample, the kit comprising at least one peptide according to theinvention or a variant thereof or a combination thereof.

According to a further aspect, the invention relates to a kit forcarrying out a method according to the invention.

The kit according to the invention comprises at least one polypeptideaccording to the invention, a variant thereof or a combination thereoffor coupling, or already coupled to a solid matrix as solid phasesupport as referred herein. Various solid matrices can be used,including but not limited to glass, polystyrene, polypropylene,nitrocellulose, quartz, ceramic, dextran or other materials. Suitableforms of the solid matrix include beads, microparticles, nanoparticles,tubes, fabrics or plates, films, slides, wells, formed from or coatedwith these materials. Typically, the solid matrix comprises microtiterwells, such as a 96-well microtiter plate.

The fixation of the peptides according to the invention to the solidmatrix in a kit according to the invention may be carried out byadsorption or chemical coupling to a solid phase support. Any meansknown in the art for immobilizing a protein or peptide to a solidsupport can be used. The peptides according to the invention can beeither covalently or non-covalently bound to the solid matrix bytechniques such as covalent bonding via an amide or ester linkage oradsorption. Peptides can be bound using binding pairs such as biotin andavidin or antibody and antigen. After the peptides are affixed to thesolid matrix, the solid matrix can be incubated with a blocking solution(containing a blocking protein such as bovine serum albumin) to reducenon-specific adsorption of antibodies in a test sample to the supportsurface. According to one aspect, the polypeptides according to theinvention can be synthesized directly on the solid matrix of the kit ofthe invention.

According to one embodiment, when the kit comprises at least onepolypeptide according to the invention, a variant thereof or acombination thereof for coupling to a solid matrix as solid phasesupport, the kit further optionally comprises coupling reagents and/or asolid matrix for performing an immunoassay.

According to another further embodiment, the kit according to theinvention further comprises at least one rinsing reagent for washingunbound material before detection in order to avoid background noisedetection. Typically rinsing reagents comprise standard buffers known inthe art.

According to another further embodiment, the kit according to theinvention further comprises at least one control sample optionallytogether with calibration information for quantification of detectedautoantibodies.

Methods Using a Kit According to the Invention

According to another aspect, the invention provides a method fordetecting a breast or an ovarian cancer from a biological fluid sampleof a mammalian subject comprising the steps of:

-   -   (a) providing a biological fluid sample from a mammalian        subject;    -   (b) bringing the said biological fluid sample into contact with        a solid matrix where at least one peptide is bound to, wherein        the contacting is under conditions sufficient for binding an        antibody present in the said biological fluid sample to the said        at least one peptide through antigen-antibody interactions and        wherein the said at least one peptide has a sequence of amino        acids of a peptide selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ        ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:        7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and        SEQ ID NO: 12 and any variant thereof;    -   (c) Removing the biological fluid sample from the solid matrix        for removing any unbound antibody from the surface of the said        solid matrix;    -   (d) Detecting the presence of an antigen-antibody complex bound        to the said solid matrix, wherein the presence of the said        complex is indicative that the biological fluid sample contains        one or more breast or ovarian-cancer associated autoantibodies.

According to a further embodiment, is provided a method according to theinvention, wherein the said at least one peptide has a sequence of aminoacids of a peptide selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

According to another further embodiment, is provided a method accordingto the invention, wherein the said biological fluid sample is broughtinto contact with the said solid matrix under step b), where acombination of peptides or of variants thereof according to theinvention is bound to said solid matrix.

According to another further embodiment, is provided a method fordetecting a breast cancer from a biological fluid sample of a mammaliansubject wherein the said biological fluid sample is brought into contactwith the said solid matrix under step b), where a combination ofpeptides or of variants thereof is bound to the said solid matrix andwhere the combination comprises: a) at least one peptide selected fromSEQ ID NO: 9 or SEQ ID NO: 12 or a variant thereof; and b) at least onepeptide selected from SEQ ID NO: 5 or of SEQ ID NO: 10 or a variantthereof.

According to another further embodiment, is provided a method fordetecting an ovarian cancer from a biological fluid sample of amammalian subject wherein the said biological fluid sample is broughtinto contact with the said solid matrix under step b), where acombination of peptides or of variants thereof is bound to the saidsolid matrix and where the combination comprises: a) at least onepeptide selected from SEQ ID NO: 9 or SEQ ID NO: 12 or a variantthereof; and b) at least one peptide selected from SEQ ID NO: 2 or ofSEQ ID NO: 4 or a variant thereof.

According to another further embodiment, is provided a method accordingto the invention, wherein the method further comprises a step ofcomparing the signal obtained under the detection step d) with the samesignal obtained for at least one control sample, wherein the signalobtained for the said at least one control sample is collectedpreviously, simultaneously or posteriori to the detection step d) forthe said biological fluid sample.

Detection of the captured/bound antibodies under step d) by any suitablemethod known in the art for detecting captured antibodies or proteins onsurfaces such as optical detection (e.g. ELISA), mass variationdetection (e.g. surface Plasmon resonance, mass spectrometry),electrical detection (e.g. impedance spectroscopy, electrochemical)techniques.

Results of the assay may be qualitative or quantitative. The amount ofcaptured/bound antibodies associated with the solid matrix can becompared with positive and negative controls. The controls are typicallyrun concomitantly with the sample to be tested. A positive control canbe a serum or a solution containing antibodies that are immunoreactivewith at least one peptide according to the invention. A negative controlcan be a serum or solution which does not contain antibodies that areimmunoreactive with at least one peptide according to the invention. Forquantization, a calibration curve using known quantities of antibody toat least one peptide according to the invention can be generated and/orused. Antibodies for use as positive controls may be produced using all,or fragments of, the amino acid sequence of a peptide according to theinvention.

The comparison with normal healthy biological fluid samples may beachieved with different methods. According to one embodiment, it may becarried out by including a control reaction with a non-diseased bloodsample. According to another embodiment, it may be carried out byemploying a value for the concentration of the endogeneous antibody fora typical biological fluid sample from a healthy subject. Typically, thecomparison of the level of endogeneous antibody present in a sampleunder investigation may be performed with respect to a value determinedin each single testing procedure or to a predetermined value. Thepredetermined value may be determined for the testing procedure ingeneral, or alternatively, the value may be valid only for a certainbatch of testing reagents. For example, the reference value may be validfor a defined calibration period only and may be redefined uponcalibration of the testing process.

The method, the kit and uses according to the invention may be suitedfor screening purposes as well as for diagnostic purposes and may beapplied in primary diagnosis as well as in monitoring of disease courseduring or after treatment.

The invention having been described, the following examples arepresented by way of illustration, and not limitation.

EXAMPLES

The following abbreviations refer respectively to the definitions below:kDa (Kilo Dalton), μg (microgram), μL (microliter), min (minute), M(molar), sec (second), BSA (bovine serum albumin), CCC (clear cellcarcinoma), ELISA (Enzyme-linked immunosorbent assay), OD (opticaldensity).

Example 1: Peptide Synthesis

Peptides were synthesized by standard peptide solid phase synthesisprocedures known to those skilled in the art. Purity of the peptides wasat least 80%. Peptides were dissolved in mother solution in 1 mg/mL inbuffer and stored in aliquots of 200 mL at −20° C. Peptides were storedin buffer carbonate, pH 9.6.

Example 2: ELISA Immunoassay Using Peptides According to the Invention

The method according to the invention is exemplified in the form of aninverse ELISA assay as described in Example 2 where peptides accordingto the invention are adsorbed on the surface of a well plate and thepresence of endogenous antibodies in the serum from patients isinvestigated as follows:

Plates

DINEX 3010 Immolux plates (Bioconcept, Switzerland) were used. Plateswere coated with the peptides at a concentration of 10 μg/mL (100μL/well) in carbonate buffer 0.05 M, pH 9.6, 2 wells per peptide andleft incubated overnight at 4° C. 80 wells were coated with the peptidesand 1 line was coated with a mixture of human IgG pooled from normalhuman sera (Southern Biotech) at 1:1000 in carbonate buffer for positivecontrol tests and another line was left empty (no coating) to measurethe non-specific binding (background) (negative control).

Procedure

The coated wells are then washed 3 times with 250 μL/well of PBST (0.05%Tween-20 in Phosphate buffer saline 0.15M, pH 7.4) to remove the coatingsolution. Wells are then incubated for 1 hour at 37° C. with BSAdiluents/blocking solution (1% BSA in PBST, 0.1 g/L Methiolate) (300μL/well). The blocking solution is then removed and the plate is washed3 times with PBST. Human sera diluted at 1:70 in the BSAdiluents/blocking solution is added (100 μL/well). The plate is thencovered with an adhesive plastic and incubated for 2 h at 37° C. Thesolution is then removed and the plate is washed 3 times with PBST (250μL/well). Horse radish-labeled antihuman antibody is added at a dilutionof 1:1000 in the BSA diluents/blocking solution (100 μL/well) andincubated 1 h at 37° C. A substrate solution is then added to the well(100 μL/well) where coloration is obtained after about 2 min incubationand the well is further incubated for 5 min at room temperature (either2-component ABTS substrate or TMB substrate). The reaction is stopped(quenching) by addition of H₂SO₄ at 2M (100 μL/well) and incubationduring 2 min. The absorbance at 450 nm is red using a microtiter platespectrophotometer. Signal cut off value was set at <0.1 OD.

Reagents

The protein detector HRP microwell kit, Anti-Human, 54-62-10, KPL, Inc.USA was used.

Peptides

Peptides used as antigens in the present reversed ELISA assay were thoseprepared according to Example 1.

Data Treatment

Data mining (detection of patterns within the data) was performed bysoftware Matlab® from Mathworks Corp, Minitab® and SPSS® from IBM Corp.

Statistical Analysis

Statistical analysis was then performed by three different methods inparallel: Principal component analysis (PCA), Linear DiscriminantAnalysis (LDA), Hierarchical Cluster Analysis and Regression Model(Pepe, 2003, “The Statistical Evaluation of Medical Tests forClassification and Prediction”, Oxford University Press). For principalcomponent analysis, data were grouped by class (patients, benignconditions and likely healthy people). A PCA was then carried out forthe three groups of samples.

Example 3: Clinical Testing on Human Breast and Ovarian Cancer SampleSets

Samples were blood samples from healthy donors and from people sufferingeither from breast or ovarian cancer.

Ovarian Cancer

A set of serum samples from patients having clear cell carcinoma andcontrols was formed as follows:

TABLE 3 Type Number Clear Cell Carcinoma 13 (ovarian cancer) Controls 10Total: 23

Those samples were tested according to a method of the invention asdescribed in Example 2. Control unrelated peptides were added to testthe selectivity of the assay. One way ANOVA analysis is applied to theresults. The same analysis was repeated by an independent laboratory andresults are presented in FIG. 1 for 4 control samples versus 4 ovariancarcinoma. A few cases which were supposed to be ovarian cancer sampleswere showing up as control in the assay according to the invention.After further investigations into sample data and records, it appearedthat these samples in fact belonged to ovarian cancer patients inremission. Therefore, it showed that the accuracy of the assay wasindeed correct in classifying these samples as non-cancer samples.

A further set of serum samples from patients having clear cell carcinoma(ovarian cancer) and controls was formed as follows:

TABLE 4 Type Number Clear Cell Carcinoma 11 (ovarian cancer) BreastCancer (CBV) 2 Benign Ovarian Condition 10 (BOV) Benign Breast Condition5 (BBV) Control healthy women 5 (GVABB) Total: 33

Results were statistically analyzed as described in Zhou et al., 2002,Statistical Methods in diagnostic medicines, Wiley, N.Y. for determiningthe specificity and sensitivity of the assay. The robustness of theassay and method according to the invention can be summarized asfollows:

TABLE 5 Number of Number of positive samples Negative samples Total ofsamples FALSE  6 (FP)  0 (FN) 6 TRUE 12 (TP) 15 (TN) 27 Total 18 15 33

Among the false positive, 2 breast cancers and 2 benign breastconditions were detected positive. Assuming True Positive (TP), TrueNegative (TP), False Positive (FP); False Negative (FN), the specificityand sensitivity of the assay according to the invention are calculatedas follows:

-   -   Specificity=TN/(TN+FP) and    -   Sensitivity=TP/(TP+FN).        which leads to 71.4% specificity, 100% sensitivity. Therefore,        an assay according to the invention presents very high        selectivity and sensitivity values for the detection of ovarian        cancers and was able to discriminate between ovarian cancer        patients and ovarian cancer patient under remission.

Breast Cancer

A set of serum samples from patients having breast cancer (CBV), breastcysts (BBV), benign ovarian cyst (BOV) and controls was formed asindicated in Table 7 below:

TABLE 6 Type Number Breast Cancer (CBV) - “patient” 66 Benign BreastCondition (BBV) - “healthy” 54 Benign Ovarian Condition (BOV) -“healthy” 50 Control healthy women (GVABB) - “healthy” 20 Total: 190

Among the patients, 29% are younger than 50-year-old and 71% are 50-yearold or older. Those samples were tested according to a method of theinvention as described in Example 2. Control unrelated peptides(references 1-5) were added to test the selectivity of the assay. Eachmeasurement was performed in triplicate. Reproducibility of the data wasassessed by analyzing the standard deviation and the coefficient ofvariation for all triplicates. Almost all the measured coefficients ofvariation are below 10% and most are below 5%, indicating goodreproducibility. Principal component analysis (PCA) was applied to thedata, the p-values obtained by Wilcoxon test (Pepe, 2003, above) and thearea under the curve values (AUC for a receiver operating curve (ROC))are mentioned for each peptide used in the assay under Table 7 below.The lower the p-value, the better the performance of the respectivepeptide. The higher the AUC above 0.54, the more accurate is the assay.

TABLE 7 Peptide p-Value AUC SEQ ID NO: 1 9.4e−7 0.72 SEQ ID NO: 2 6.0e−50.68 SEQ ID NO: 3 6.5e−12 0.8 SEQ ID NO: 4 1.4e−9 0.77 SEQ ID NO: 54.4e−13 0.82 SEQ ID NO: 6 0.51 0.63 SEQ ID NO: 7 1.1e−5 0.69 SEQ ID NO:8 0.27 0.55 SEQ ID NO: 9 0.071 0.58 SEQ ID NO: 10 0.0036 0.63 SEQ ID NO:11 1.9e−5 0.69 SEQ ID NO: 12 4.2e−6 0.7 Reference 1 0.64 0.52 Reference2 0.77 0.51 Reference 3 0.95 0.5

Results were further analyzed using Linear Discriminant Analysis (LDA).The patient data was split in two equal parts, one consisting in alearning set (used to train the classification algorithm), and the otherone being a testing set (used to assess the performance of theclassifier). Results are presented under FIG. 2. The score obtained withthe LDA procedure was used to classify the samples from the testing set.Scores are indicated for cases vs controls (2A), by subgroup (2B), andin the form of a Receiver Operating Characteristic (ROC) curve (2C). TheArea Under the Curve (AUC) obtained was 0.92, an excellent value.

A numerical summary of the results obtained for different computedthresholds (1 to 21) is shown in the Table 8 below. The thresholds werechosen uniformly within the range of possible scores (in the range −5 to+5); FP, TP, TN and FN columns contain the respective number of FalsePositives, True Positives, True Negatives and False Negatives for thegiven threshold; the sum of these four numbers is always 95 (the totalnumber of samples in the testing set). The specificity and sensitivitygiven by TN/TN+FP and TP/TP+FN measure the accuracy of the thresholdwith regards to the total of positive and negative samples in thetesting set. The Positive Predictive Value (PPV) and the NegativePredictive Value (NPV) indicate the proportion of patients testedpositive (respectively negative) which are correctly diagnosed, and arecalculated as TP/TP+FP and TN/TN+FN.

TABLE 8 Threshold FP TP TN FN Spec. Sens. PPV NPV 1 −5.35 62 33 0 0 0.001.00 0.35 — 2 −4.99 61 33 1 0 0.02 1.00 0.35 1.00 3 −4.28 60 33 2 0 0.031.00 0.35 1.00 4 −3.58 59 33 3 0 0.05 1.00 0.36 1.00 5 −2.52 57 33 5 00.08 1.00 0.37 1.00 6 −2.16 52 33 10 0 0.16 1.00 0.39 1.00 7 −1.81 47 3315 0 0.24 1.00 0.41 1.00 8 −1.46 43 33 19 0 0.31 1.00 0.43 1.00 9 −1.1039 33 23 0 0.37 1.00 0.46 1.00 10 −0.75 32 33 30 0 0.48 1.00 0.51 1.0011 −0.40 24 32 38 1 0.61 0.97 0.57 0.97 12 −0.04 21 30 41 3 0.66 0.910.59 0.93 13 0.31 11 29 51 4 0.82 0.88 0.72 0.93 14 0.66 8 29 54 4 0.870.88 0.78 0.93 15 1.02 5 24 57 9 0.92 0.73 0.83 0.86 16 1.37 2 18 60 150.97 0.55 0.90 0.80 17 1.72 2 11 60 22 0.97 0.33 0.85 0.73 18 2.08 1 461 29 0.98 0.12 0.80 0.68 19 2.43 1 1 61 32 0.98 0.03 0.50 0.66 20 3.140 1 62 32 1.00 0.03 1.00 0.66 21 5.26 0 0 62 33 1.00 0.00 — 0.65

In brief, the value measured for each peptide is multiplied by a certainweight, the sum of all these weighted values give the resulting score.As an illustration of this model, “sample A” of breast cancer has ascore of 1.29. Table 9 below shows the weights obtained from theselected LDA model as described above for 8 peptides according to theinvention and corresponding measurements from those peptides for thissample A. Applying the weights to the eight measurements yields thefollowing results:

TABLE 9 SEQ ID NO: 1 2 3 4 5 6 9 10 Measure- 0.59 0.49 0.23 0.50 0.320.34 0.60 0.38 ment Weights −1.71 13.11 0.94 −5.89 1.05 −11.86 5.83−3.37 Combina- −1.00 6.47 0.22 −2.94 0.33 −3.98 3.47 −1.26 tion

The sum of the combined values indeed yields a score of 1.29 (except forrounding errors).

Results for all ovarian samples and controls were statistically analyzedas described in Zhou et al., 2002, Statistical Methods in diagnosticmedicines, Wiley, N.Y. for determining the specificity and sensitivityof the assay. The robustness of the assay and method according to theinvention can be summarized as follows: 87.3% specificity, 80.3%sensitivity, 74.7% positive prediction value, 90.5% negative predictionvalue.

Therefore, an assay according to the invention presents very highselectivity and sensitivity values for the detection of breast cancer.

The invention claimed is:
 1. A kit for detecting one or moreautoantibodies associated with a breast cancer or an ovarian cancer in abiological fluid sample of a mammalian subject, the kit comprising apeptide or a combination of peptides coupled to a solid support matrix,wherein the peptide or combination of peptides comprises: a) a peptideconsisting of the amino acid sequence set forth in SEQ ID NO: 5; b) acombination of peptides comprising a peptide consisting of the aminoacid sequence set forth in SEQ ID NO: 5 and a peptide consisting of theamino acid sequence set forth in SEQ ID NO: 1, a peptide consisting ofthe amino acid sequence set forth in SEQ ID NO: 4 and a peptideconsisting of the amino acid sequence set forth in SEQ ID NO: 12; or c)a combination of peptides comprising 1) a peptide consisting of theamino acid sequence set forth in SEQ ID NO: 5 and 2) at least onepeptide consisting of the amino acid set forth in SEQ ID NO: 1 SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or acombination thereof, wherein the one or more autoantibodies bind to oneor more of the peptides comprised in the kit.
 2. The kit of claim 1,wherein the peptide is a peptide consisting of the amino acid sequenceset forth in SEQ ID NO:
 5. 3. The kit of claim 1, wherein said kitcomprises a combination of peptides comprising a) a peptide consistingof the amino acid sequence set forth in SEQ ID NO: 5; and b) at leastone peptide consisting of the amino acid sequence set forth in SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12 or a combination thereof.
 4. The kit of claim 1, wherein said kitcomprises a combination of peptides comprising a peptide consisting ofthe amino acid sequence set forth in SEQ ID NO: 5, a peptide consistingof the amino acid sequence set forth in SEQ ID NO: 1, a peptideconsisting of the amino acid sequence set forth in SEQ ID NO: 4 and apeptide consisting of the amino acid sequence set forth in SEQ ID NO:12.
 5. A method for detecting a breast or an ovarian cancer from abiological fluid sample of a mammalian subject comprising the steps of:(a) providing a biological fluid sample from a mammalian subject; (b)bringing said biological fluid sample into contact with the solid matrixof the kit of claim 1, wherein the contacting is under conditionssufficient for binding an antibody present in said biological fluidsample to said solid matrix through antigen-antibody interactions; (c)removing the biological fluid sample from the solid matrix to removeunbound antibody from the surface of said solid matrix; and (d)detecting the presence of an antigen-antibody complex bound to saidsolid matrix, wherein the presence of said complex is indicative thatthe biological fluid sample contains one or more breast orovarian-cancer associated-endogenous antibodies.
 6. The method accordingto claim 5, wherein the peptide bound to the solid matrix is a peptideconsisting of the amino acid sequence set forth in SEQ ID NO:
 5. 7. Themethod according to claim 5, wherein the combination of peptides boundto the solid matrix comprises a peptide consisting of the amino acidsequence set forth in SEQ ID NO: 5 and a peptide consisting of the aminoacid sequence set forth in SEQ ID NO: 1, a peptide consisting of theamino acid sequence set forth in SEQ ID NO: 4 and a peptide consistingof the amino acid sequence set forth in SEQ ID NO:
 12. 8. The methodaccording to claim 5, wherein the combination of peptides bound to thesolid matrix comprises 1) a peptide consisting of the amino acidsequence set forth in SEQ ID NO: 5 and 2) at least one peptideconsisting of the amino acid set forth in SEQ ID NO: 1 SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or a combinationthereof.